Program Director/Principal Investigator (Last, First, Middle): Okhuysen, Pablo C/Chappell, Cynthia L Title: MUCOSAL DETERMINANTS OF CRYPTOSPORIDIUM INFECTION PI: Pablo C. Okhuysen, MD MD Anderson Cancer Center PI (subcontract): Cynthia L. Chappell, PhD UT School of Public Health PROJECT SUMMARY Cryptosporidium causes diarrheal illness and is second only to Rotavirus in prevalence among children in developing areas. This environmentally resilient, highly infectious NIAID category B agent is also a major cause of waterborne outbreaks of diarrhea in the US. In malnourished children, the elderly and in immunosuppressed individuals, infection causes chronic debilitating illness that can be fatal. The parasite cannot be grown efficiently in vitro and no effective treatment is available. New approaches to study the pathophysiology of this agent are needed in order to develop strategies for treatment and control. We have previously shown that when exposed to the parasite, healthy adult volunteers demonstrate distinct outcomes that are dependent on parasite genotype/species. The molecular basis for these differences are unknown. We also recently showed that when exposed to the parasite, volunteers with high levels of intestinal indole (IND), a gut bacterial product, are protected from infection but nothing is known about why this association exists or its mechanism of action. Thus, the overarching goal of this proposal is to examine potential mechanisms for the observed protective phenomenon in the context of parasite genome diversity. To this end, we will use new techniques, such as full length (FL) genome sequencing and organoid cultures, to examine the effect of microbiome composition and IND compounds (ICs) on parasite infectivity in cell lines and intestinal organoid cultures. Genomic sequencing and/or RNA expression of the host and/or parasite will help to delineate essential components of the host- parasite interaction thereby suggesting potential interventions. Specifically, we will study the microbiome composition and generate FL genomes from stool samples of: adults previously challenged with Cryptosporidium; HIV-infected individuals; and immunosuppressed cancer patients, the latter two groups with community-acquired Cryptosporidium. We will sequence parasite genomes and test the direct effects of ICs on the parasite infectivity in cell lines and organoids by monitoring parasite binding, invasion, and replication. We will also measure expression of cytokines and other immunomodulatory factors produced by infected cells to better understand the role of the immune response in Cryptosporidium pathogenesis. Finally, in a proof of concept experiment, we will deprive adult mice of IND, then challenge them with Cryptosporidium, and monitor for infection. Metagenomic sequencing will monitor changes in the bacterial communities before and after manipulation. Taken together, we expect these experiments will shed light on the mechanism involved in IND- associated protection from genotypically diverse Cryptosporidium infections. We postulate that IND and/or Cryptosporidium infection upregulate host cell expression and secretion of cytokines via the aryl hydrocarbon receptor pathway, which is involved in the immune response. We expect these studies will lay the foundation for developing new treatment modalities, such as IND or ICs, or possibly the use of IND-producing bacteria as probiotics to increase gut indole levels, thus promoting a gut environment which would make individuals less susceptible to cryptosporidiosis. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page